It has recently become appreciated that ribosomal peptide natural products (RiPPs) represent one of the major groups of bioactive natural products on Earth. The compounds are found nearly universally in bacteria, where they place diverse roles including quorum signaling, cofactor synthesis, and chemical defense. Most commonly, RiPPs exhibit potent antibiotic activity. They are key players in modern drug discovery and development. Perhaps more importantly, RiPPs present an enormous storehouse of posttranslational machinery that can be used to design drug-like compounds. They are readily manipulated for the rational engineering and optimization of desired properties. In this proposal, we will use this diverse RiPP biosynthetic machinery to better understand posttranslational enzymes, to optimize and engineer production platforms, and to develop technologies to better harness these complex pathways in the design and discovery of new antibiotics. Our specific aims are to: 1) Characterize unique RiPP posttranslational enzymes using chemical, biochemical, and genetic methods; 2) Improve compound production and analog synthesis in heterologous hosts; 3) Discover new antibiotics by implementing novel bioassays.